Are you tired of running Western blots? Why not consider dot blots?
For this week's Antibody Application series, Deric Griffin will tell us about the principles of dot blots and some bench tips.
Deric recently completed his PhD in the Translational Biology and Molecular Medicine program at Baylor College of Medicine (Congratulations!).
Take it away Deric!
What is Dot Blot?
Dot blot is a simple way to test for the presence of a protein of interest (POI) in a sample. The technique is actually very similar to the Western blot, but dot blot, for reasons we’ll cover later, is a faster, cheaper, and easier technique. As an aside, the dot blot can also be used for detection of nucleic acids, but for the sake of simplicity, we’ll restrict this to a discussion of protein.
How Does it Work and What Would You Use it For?
Dot blot relies on the same principle that many immunological techniques rely on: the recognition and binding of an antigen by an antibody. Briefly, dot blot utilizes a dry nitrocellulose or PVDF membrane that has been "dotted" with sample homogenate (typically a sample volume of ~2uL/dot).
The membrane is then blocked for non-specific binding using a blocking buffer (I use 5% BSA in TBS-T), followed by incubation with a primary antibody specific to the POI for 30 mins to 1 hour at room temperature. This step is then followed by incubation with a secondary antibody (also at room temperature for 30 min to 1 hour) that allows visual detection and quantification of the target protein through methods such as chemiluminescence or fluorescence. Our lab uses the fluorescence based Odyssey system by Licor for our blots.
This particular technique is usually used for detection and quantification. The technique also provides a quick way to determine if an antibody is non-specific, particularly for a secondary antibody.
If They Are So Similar, Then Why Not Just Run a Western?
Ah, the big question! A Western blot can give you the same information as a dot blot, but Westerns do have relative drawbacks:
More materials: A Western requires the same materials as a dot blot, in addition to (at a minimum) a polyacrylamide gel, molecular weight ladder, loading dye, along with apparatuses and buffers for running the gel and transferring the protein to a protein sensitive membrane.
More steps: You’ll not only have to load and run the sample on a polyacrylamide gel (which you’ll have to make yourself or purchase a pre-cast gel), but also transfer the sample from the gel to a membrane.
More time: Perhaps the biggest drawback. As a bench researcher, you tend to count your time in the number of assays and experiments you can run that day. A dot blot, from start to finish, can be done within ~3 hours. A Western can take up to two days of work when including the transfer step and a possible overnight incubation with the antibody.
Limited samples: The number of samples assessed in a Western is limited by the number of lanes in the polyacrylamide gel. A dot blot, by comparison, can screen a large number of samples for presence of a POI at once.
More troubleshooting: As the Western blot has more steps, there are more chances for something to go wrong and also more individual steps to investigate.
What Are Some Limitations for Dot Blot?
The biggest drawback to the dot blot is that it cannot separate proteins by size. While a noticeable drawback in itself, this can actually affect other aspects:
Signal normalization: Dot blots do not typically use housekeeping proteins to normalize signal.
Higher background: As protein in the sample has not been separated by size, this can make detection of a false positive (such as non-specific binding to peptide fragments) difficult to detect.
Multiple targets: A dot blot would not allow for comparison of a normal and modified target within the same blot.
In summary, the dot blot, while not as informative as techniques such as the Western blot, is an inexpensive, quick and useful technique for antibody comparison and assessing a large number of samples. If simple detection of POI presence is your goal, you should definitely make the dot blot a go-to technique. A nice general protocol is available from Abcam.
In my next article, I'd like to share with you some bench tips I've learned for dot blot throughout my PhD. Stay tuned!
For a quick and comprehensive review of published dot blot data for better antibody selection, check out BenchSci:
